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01.2 chiorazzi pc  aecc_retreat_5-5-10_for_website
 

01.2 chiorazzi pc aecc_retreat_5-5-10_for_website

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  • I would like to thank the organizers for giving me the opportunity to discuss our research results with you. This represents the work of not only myself but of many others including… Lu, Katerina, Rosa Steve and Kanti for clinical collaboration Nick for support, guidance and inspiration and others that I will acknowledge throughout the talk
  • This is a typical example of flow cytometry data showing that CLL 068 mAb reacts to MEACs. This is a negative control. This shows that CLL 068 binds to a subset of dead cells (AV-PE positive) and not live cells. This shows that CLL 068 binds only to MEACs (MYHIIA positive). And not other cells.
  • This is a graph that shows the binding of 26 CLL mAbs listed on the x-axis to MEACs… … with the cutoff of 1.5 for MEAC binding. 16 mAbs bind well/. This is a grouping of the CLL mAbs based on having a shared common “stereotyped” sequence, which I do not have time to describe, … but would just like to point out that the stereotyped groups bind in the same manner. This shows which CLL mAbs are mutated or unmutated, … U = is less than 2% mutation in the IGHV, which correlates with bad patient survival … M = is greater than 2% mutation in the IGHV, which correlates with good patient outcome. All the MEAC binding mAbs are UM, except for 1 (15/16), which binds like the same stereotype subgroup “1” and is a borderline “Mut”. Those mAbs that do not bind MEACs well are a mixture of Mut (6) and four UM (4) mAbs. Because survival correlates with IGHV mutation, perhaps MEAC binding correlates with survival and help distinguish the UM that may survive better.. .
  • Indeed binding well to MEACs correlates to poor patient survival (24 patients have survival data)… Hi binding has a median survival of 99 months (n=15), Whereas Lo binding has not reached median survival (n=9). This is statistically significant P<0.06) in this patient cohort. … this is because one mutated (CLL 154) and two unmutated (CLL 376 and 412) IGHV CLL patients having survival outcomes contrary to that expected for their IGHV mutation status.
  • IGHV mutation status versus patient survival (24 patients have survival data)… UM IGHV has a median survival of 118 months (n=18), Whereas Mut IGHV has not reached median survival (n=6). This is not quite statistically significant P<0.06. This significance is not as good as MEAC binding. … this is because one mutated (CLL 154) and two unmutated (CLL 376 and 412) IGHV CLL patients having survival outcomes contrary to that expected for their IGHV mutation status.
  • This is a graph that shows the binding of 26 CLL mAbs listed on the x-axis to MEACs… … with the cutoff of 1.5 for MEAC binding. 16 mAbs bind well/. This is a grouping of the CLL mAbs based on having a shared common “stereotyped” sequence, which I do not have time to describe, … but would just like to point out that the stereotyped groups bind in the same manner. This shows which CLL mAbs are mutated or unmutated, … U = is less than 2% mutation in the IGHV, which correlates with bad patient survival … M = is greater than 2% mutation in the IGHV, which correlates with good patient outcome. All the MEAC binding mAbs are UM, except for 1 (15/16), which binds like the same stereotype subgroup “1” and is a borderline “Mut”. Those mAbs that do not bind MEACs well are a mixture of Mut (6) and four UM (4) mAbs. Because survival correlates with IGHV mutation, perhaps MEAC binding correlates with survival and help distinguish the UM that may survive better.. .
  • These results lead to the following inferences…
  • Just to end with a MODEL cell death (CLL or other) leading to MEAC formation and exposure of MYHIIA and other epitopes observed by Rosa This could be important for the initiation of CLL or the ongoing stimulation that may be required for CLL growth and development.

01.2 chiorazzi pc  aecc_retreat_5-5-10_for_website 01.2 chiorazzi pc aecc_retreat_5-5-10_for_website Presentation Transcript

  • Immunobiology of chronic lymphocytic leukemia Nicholas Chiorazzi Departments of Cell Biology and of Medicine Albert Einstein College of Medicine and The Feinstein Institute for Medical Research North Shore – LIJ Health System
  • Ig V gene sequencing Autoantigen reactivity Rajendra Damle Charles Chu Joy Yan Rosa Catera Bradley T. Messmer Manuela Woelfle Emilia Albesiano Katerina Hatzi Angelo Valetto Zev Sthoeger Fabio Ghiotto Eric Meffre Franco Fais Maxime Herve Gregg Silverman Collaborators Kanti R. Rai Steven L. Allen Jonathan E. Kolitz Matthew Kaufman Manlio Ferrarini
  • Chronic Lymphocytic Leukemia Most prevalent adult leukemia of the Western world  15,340 new cases and 4,500 deaths in 2007 Disease of aging individuals (>50) with incidence increasing with each subsequent decade Usually affects men > women; Caucasian > African > Asian
  • Clinical dilemma Clinical courses of patients are heterogeneous and that of an individual patient is unpredictable  Some patients follow very benign courses living decades and dying with the disease, not from it  Other patients follow more malignant courses living only a few years after diagnosis, despite therapy CLL remains an incurable disease Therefore, “wait and watch” / “wait and worry” approaches are taken by clinician and patient
  • Chronic Lymphocytic Leukemia Lymphocytosis seen in blood, but most leukemic cells are in non-vascular areas including bone marrow, lymph nodes, and spleen Usually detected upon routine blood workup as an elevated white cell or lymphocyte count (~3x1010 total in blood) Clonal expansion of a CD5+ B lymphocyte with low surface immunoglobulin (Ig) Most clones express predominantly IgM isotype surface Ig, but ~7% are predominantly IgG or IgA, though in those cases IgM clonal relatives can be found
  • Clonal disease of B lymphocytes CD19 CD5 CD23 Smudge cell smIg (BCR) CLL cells Granulocyte
  • Take home messages 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid structure 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by apoptosis and other catabolic processes 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical disease
  • Take home messages 1. CLL results from the non-random selection and transformation of B lymphocytes expressing B-cell antigen receptors (BCRs) of restricted amino acid structure
  • Ig molecules and their genes NH2 VK CK VH Fab CDR3 CDR2 CDR1 CH1 Fc FR4 FR3 FR2 FR1 VH D JH CH -S - S- Hinge CH2 Region JH D VH Chromosome 14 VK JH CK CH3 Chromosome 2 COOH JK VK Ig Molecule V Region Ig Genes
  • CLL cells differ from normal CD5+ B cells by the overuse of certain autoreactive genes Fais et al. J Clin Invest 98: 1659, 1998
  • CLL clones differ in the degree of somatic mutations, especially in particular IgV genes VH Specific % Cases with Family VH Gene Mutations All cases - 50.7 1 - 33.3 1-69 10.0 3 - 66.7 3-07 90.0 4 - 41.2 4-34 55.0 Fais et al. J Clin Invest 98: 1659, 1998
  • Ig VH gene mutation status of CLL cells is an important prognostic indicator of outcome ≥ 2% mutation ≥ 2% mutation < 2% mutation < 2% mutation Damle et al. Hamblin et al. Blood 94: 1840, 1999 Blood 94: 1848, 1999
  • Ig molecules and their genes NH2 VK CK VH Fab CDR3 CDR2 CDR1 CH1 Fc FR4 FR3 FR2 FR1 VH D JH CH -S - S- Hinge CH2 Region JH D VH Chromosome 14 VK JH CK CH3 Chromosome 2 COOH JK VK Ig Molecule V Region Ig Genes
  • CLL clones are culled from the normal B-cell repertoire based on structural constraints of the B-cell antigen receptor
  • IgV gene segment recombination Heavy Chain Light Chain (k/λ) VH (44) D (27) JH (6) VL (46/36) JL (5/7) = RAG mediated recombination HC = VH x D x JH = 44 x 27 x 6 = 7,128 VHDJH rearrangement: ~1 : 7,000
  • ~1% of CLL patients express a BCR with a VH 1-69 gene exhibiting very similar HCDR3s often comprised of the same VH-D-JH segments Widhopf et al. Blood 104: 2499-2504, 2004
  • Ig V region gene segment recombination Heavy Chain Light Chain (k/λ) VH (44) D (27) JH (6) VL (46/36) JL (5/7) = RAG mediated recombination HC = VH x D x JH = 44 x 27 x 6 = 7,128 k = Vk x Jk = 46 x 5 = 230 VHDJH / VLJL rearrangement: ~1 : 3 x 106 l = Vl x Jl = 36 x 7 = 252
  • CLL cases with remarkably similar B-cell receptors V Gene Germline Percent D J CLL Family V Gene Difference Gene Gene CLL 039 VH4 4-39 0.3 6-13 5b VkI O12 0.0 - k2 CLL 057 VH4 4-39 0.7 6-13 5b VkI O12 0.0 - k1 CLL 114 VH4 4-39 0.3 6-13 5b VkI O12 0.0 - k1 CLL 202 VH4 4-39 0.7 6-13 5b VkI O12 0.0 - k1 CLL 209 VH4 4-39 0.3 6-13 5b ? ? ? - ? Ghiotto et al. J Clin Invest 113: 1008, 2004
  • IgV gene segment recombination Heavy Chain Light Chain (k/λ) VH (44) D (27) JH (6) VL (46/36) JL (5/7) = RAG mediated recombination
  • Because of the differences that occur at the junctions when gene segments combine, the likelihood that the same VHDJH- VLJL rearrangement with the same junctional characteristic would occur in two different B cells is even much more remote ≈ 1 / 1x108 – 1 : 1x1012 Therefore, if the gene structure of the Ig variable region found in B-CLL cells from different patients is very similar or identical, then this must indicate a selective process of leukemogenesis that targets B cells with a given type of Ig V region.
  • Heavy chain sequence alignment VH1-69 D3-16 JH 3 CAR YYDYVWGSYRY DAFDIWG CLL068: CAR GG DYDYVWGSYRS N DAFDIWG CLL258: CAR GG IYDYVWGSYRP N DAFDIWG CLL022: CAR GG DYDYVWGSYRP N DAFDIWG CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG aCLA*: CAR GG NYDYIWGSYRS N DAFDIWG  Natural autoantibody *aCLA = anti-cardiolipin ab Messmer et al. J Exp Med 2004; 200: 519-525
  • Almost 30% of patients with chronic lymphocytic leukemia carry stereotyped receptors Stamatopoulos et al. Blood 109:259-270, 2007 Murray et al. Blood 111:1524-1533, 2008 >35% chance of fitting into a stereotypic set if U-CLL or if express a specific VH gene (1-69, 3-21, 4-39) associated with poor outcome
  • Take home messages 2. These BCRs can be poly- and auto-reactive, binding natural as well as novel autoantigens generated by apoptosis and other catabolic processes
  • Expression of recombinant CLL mAbs Transfection of 293T HEK cells using Lipofectamine 2000 1. Plasmid DNA 2.Lipofectamine 3. Lipofectamine 2000 4. Liposomes are added in carrying heavy and 2000 Reagent Reagent and DNA are 293T HEK cell culture light chain Ig gene mixed and incubated Immuno assay for quantification of CLL mAb 4-5 days of culture 293T HEK cell line Antibody purification using Protein G beads Wardemann et al. Science 301:1374, 2003
  • Herve et al. J Clin Invest 115:1636-1643, 2005
  • Reactivity of recombinant mAbs with viable HEp-2 cells that were permeabilized to allow Ab entry [“Anti-cell antibodies”] ~20% of mAbs from CD5+ B cells from normal subjects ~75% of mAbs from CLL cells  ~90% from unmutated CLL  ~60% from mutated CLL Reactivity mainly with cytoplasmic structures and occasionally with nucleoli. Only one mAb from M-CLL patient reacted with nucleus in a homogeneous pattern Herve et al. J Clin Invest 115:1636-1643, 2005
  • Autoreactivities of individual CLL BCRs/mAbs 1. VH1-69/D3-16/JH3 + VK3-20: Non-muscle myosin heavy chain IIA (Chu et al. Blood 112: 5122-5129, 2008) 2. VH4-39/D6-13/JH5 + VK1D-39/JK1 Vimentin (Chu et al. Blood 112: 5122-5129, 2008) 3. 60% of U-CLL and 10% of M-CLL: Apoptosis-associated autoantigens (Catera et al. Mol Med 2008; 14: 665-674 )
  • Herve et al. J Clin Invest 115:1636-1643, 2005
  • Heavy chain sequence alignment VH1-69 D3-16 JH 3 CAR YYDYVWGSYRY DAFDIWG CLL068: CAR GG DYDYVWGSYRS N DAFDIWG CLL258: CAR GG IYDYVWGSYRP N DAFDIWG CLL022: CAR GG DYDYVWGSYRP N DAFDIWG CLLSMI: CAR GG NYDYIWGSYRS N DAFDIWG aCLA*: CAR GG NYDYIWGSYRS N DAFDIWG  Natural autoantibody *aCLA = anti-cardiolipin ab Messmer et al. J Exp Med 2004; 200: 519-525
  • mAb 068 binds 225KDa molecule C. Chu et al. Blood 112: 5122-5129, 2008
  • LC MS/MS identifies 225KDa band as non-muscle myosin heavy chain IIA (MYHIIA) C. Chu et al. Blood 112: 5122-5129, 2008
  • CLL mAb 068 co-localizes with pAbs to MYHIIA C. Chu et al. Blood 112: 5122-5129, 2008
  • Autoreactivities of individual CLL BCRs/mAbs 1. VH1-69/D3-16/JH3 + VK3-20: Non-muscle myosin heavy chain IIA (Chu et al. Blood 112: 5122-5129, 2008) 2. VH4-39/D6-13/JH5 + VK1D-39/JK1 Vimentin (Chu et al. Blood 112: 5122-5129, 2008) 3. 60% of U-CLL and 10% of M-CLL: Apoptosis-associated autoantigens (Catera et al. Mol Med 2008; 14: 665-674 )
  • CLL mAbs react with apoptotic (not healthy) cells A B 3.70 28.17 16.77 13.86 Annexin V RAMOS 67.27 0.92 69.14 0.23 CLL014 DO13 C D 52.86 18.12 55.35 16.96 Annexin V Jurkat 28.47 0.55 27.37 0.32 CLL014 DO13 R. Catera et al. Mol Med 14: 665-674, 2008
  • Apoptotic B and T cells are a source of autoantigens for CLL mAbs SUMMARY of the results: - More than 60% (18/28) of the mAbs tested reacted with these two cell types - 15 of the 18 reactive mAbs used an unmutated VH gene, only 3 used a mutated VH gene R. Catera et al. Mol Med 14: 665-674, 2008
  • Antigens bound at the surface of apoptotic cells have translocated from intracellular compartments Cytox Orange Annexin V CLL 114 Merge Membrane blebs Apoptotic body without DNA Apoptotic body with DNA R. Catera et al. Mol Med 14: 665-674, 2008
  • MYHIIA is one of the intracellular antigens that translocates to the surface and is bound by CLL mAbs
  • MEAC: MYHIIA exposed apoptotic cell Late apoptotic Live Early apoptotic Chu et al. Blood 2010 in press
  • CLL 068 mAb binds to MEACs Negative Apoptotic MEACs Chu et al. Blood 2010 in press
  • Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al. Blood 2010 in press
  • Take home messages 3. The clinically-distinct subgroups differ in the retention or loss of poly- and auto-reactivity, with the retention of polyreactivity being associated with worse clinical disease
  • Polyreactivity is a feature primarily of unmutated CLL cells Herve et al. J Clin Invest 115:1636-1643, 2005
  • Ig VH gene mutation status of CLL cells is an important prognostic indicator of outcome ≥ 2% mutation ≥ 2% mutation < 2% mutation < 2% mutation Damle et al. Hamblin et al. Blood 1999; 94: 1840 Blood 1999; 94: 1848
  • Binding well to MEACs correlates with poor patient survival Lo binding ?? Months (n = 9) Hi binding 99 months (n = 15) Chu et al. Blood 2010 in press
  • In this limited series, MEAC binding correlates better with patient survival than IGHV mutation status Mutated ?? Months (n = 6) Unmutated 118 months (n = 18) Chu et al. Blood 2010 in press
  • Many CLL mAbs bind MEACs MEAC binding Subset Mutation Chu et al. Blood 2010 in press
  • Inferences 1. MEACs may be a source of autoantigens driving CLL disease 2. The origin of many CLL clones may be cells that produce natural antibodies to MEACs or other natural products of catabolism  B1-like cells, MZ B cells? 3. If MEAC binding is a “better” predictor of patient survival than IGHV mutations status, is it because the former implies antigen-binding activity whereas the latter directly measures it?
  • B-CLL Initiation M-CLL evolution U-CLL hypothesis Progression MEACs MYHIIA+ Vimentin Filamin B Oxidation Chemical Modification